Novel agents for ameliorating motor disorder

ABSTRACT

A novel medicament for treating neurodegenerative diseases, especially for ameliorating dyskinesia, comprising as an active ingredient selenoprotein P and/or a peptide fragment or a series of peptide fragments derived from said protein is provided. The novel medicament for treating neurodegenerative diseases, especially for ameliorating dyskinesia, according to the present invention is suitably applicable to diseases with decrease in motor function.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel use of plasma proteins,belonging to the field of medical drugs. More specifically, the presentinvention relates to a medicament for treating neurodegenerations causedby cell death or cellular degeneration of the cells consisting of thenervous system, such as aging, trauma, cerebrovascular disorders,immunopathy, ataxia, epilepsy, motor neuron disorders, and the like.Still more specifically, the present invention relates to a medicamentfor treating neurodegenerative disorders, especially for dyskinesia,comprising as an active ingredient selenoprotein P, one of plasmaproteins, more preferably a peptide fragment or a series of peptidefragments derived from the C-terminal of selenoprotein P.

BACKGROUND OF THE INVENTION

Neural diseases, where cell death or cellular degeneration of the cellsconsisting of the nervous system such as neurocytes or glia cells areinvolved, include cerebrovascular disorders including cerebralinfarction, cerebral hemorrhage, subarachnoid hemorrhage, multi-infarctdementia, Binswanger type leukoencephalopathy, and chronic subduralhematoma; autoimmune diseases including multiple sclerosis,Guillain-Barré syndrome, and collagen disease; neurodegenerativediseases including spinocerebellar degeneration, Shy-Drager syndrome,amyotrophic lateral sclerosis, Alzheimer disease, Pick disease,Huntington chorea, Parkinson disease, progressive supranuclear palsy,epilepsy, and Prion disease; dementia or dysbasia associated with aging,or traumatic injury of spinal chord or cerebral disorders due to trafficaccident. Among most of these diseases is commonly observed decrease inmotor function (dyskinesia).

Dyskinesia includes muscular disorders, neural disorders, and disordersin bones and joints. Among these, central neural disorders leading todyskinesia is classified according to the regions suffered into cerebral(frontal lobe), cerebellar, vestibular (labyrinth), and spinaldyskinesia.

Cerebral dyskinesia is caused by disorder in cerebral cortex, especiallythe frontal lobe, and can be observed in case of cerebrovascular lesion,cerebral atrophy, trauma, tumor, Pick disease, and chronic subduralhematoma. It exhibits atactic abasia and decrease in mental function.

Cerebellar dyskinesia is a significant symptom associated with, forinstance, cerebellar disorders such as cerebellar tumor, vasculardisorders, degenerative disorders, cerebellar atrophy, or deformity.Lesions in the vermis induce trunk ataxia, exhibits astasia-abasia, andgluteus maximus gait, yields difficulty in maintaining posture andposition with disorder in balance. On the other hand, disorders in thecerebellar hemisphere exhibit abnormality of tonus in limb muscles anddecrease in myotony and are accompanied by maldispositional gait towardsthe affected lateral direction, incoordination, wrong indication infinger-finger test or finger-nose test, dysmetria, Holmes-Stewartphenomenon, as well as intention tremor and cerebellar speech (scanning,explosive).

Vestibular (labyrinth) ataxia is caused by vestibular malfunctions andmost of its cause is supposed to be the presence of, or sequela from,otological disorders in the internal ear, including, for instance,Meniere disease, sudden deafness, disorders in the balance-relatednerves due to drug poisoning such as streptomycin or kanamycin, trauma,syphilis, acoustic trauma hearing loss, otosclerosis, and otitis interna(and its sequela).

In case of spinal dyskinesia, also called ataxia of posterior funiculus,disorders in posterior column of spinal cord lead to disorders inbathyesthesia, i.e. positional sensibility, articular sensibility andsensibility of grasp, resulting in ataxia. It is markedly observed inFriedreich's ataxia, subacute combined degeneration of spinal cord,locomotor ataxia, and the like.

These neurodegenerative diseases with dyskinesia are deeply related withcell death or cell degeneration of the neurocytes in their pathologicalconditions. For example, in case of poly(glutamic acid) disease,observed in Huntington disease, spinocerebellar degeneration(Machado-Joseph disease, Friedreich's ataxia, etc.) or myotonicdystrophy, or Alzheimer disease, cell death or cell degeneration of theneurocytes due to intracellular accumulation of abnormal proteins isobserved. For motor neuronal disorders, typically amyotrophic lateralsclerosis, its cause is thought to be cell death of the neurocytes dueto generation of free radicals or excessive accumulation of glutamicacid, increase in intracellular calcium ion level, or generation of NO.In case of Parkinson disease, its principal cause is thought to bedegeneration of dopaminergic neurocytes in the stratum compacta ofsubstantia nigra.

As the aging process proceeds, man's physical function continuallydeteriorates. As a morphological basis of the aging process, organsexhibit atrophy (i.e. decrease in weight). For example, the proceedingsof the aging process render the brain atrophic, in its extremityresulting in gait disability or dementia. From histopathological pointof view, there are observed degeneration or drop-off of the neurocytes,senile plaques, or change in Alzheimer fibril. Cell death or cellulardegeneration of the nervous system is supposed to be induced byoxidative stress such as free radicals or glycation. It is also reportedthat biochemical observation of the brain autopsy revealed decrease inneurotransmitters, especially disturbance of cholinergic neurons, butthe cause still remains to be elucidated.

Even if under healthy conditions, a man can fall into gait disabilitythrough injury of the spinal cord or the brain by, for instance, trafficaccident. When a strong external pressure is applied in case ofaccidents, dislocation fracture of the spinal column occurs to press thespinal cord or to cause fractural injury in the spinal cord, resultingin injury of the spinal cord. Other causes include radiation burn,incised wound, or stab wound. It can also occur in case where wound isunobservable in bone as is often seen in hyperextension injury of thecervical vertebrae in which detrition and pressure lesion of the spinalcord is observed with marked hemorrhage and edema in parenchyma of thespinal cord. Its clinical symptom includes incomplete or completesystemic paralysis.

Autoimmune diseases are also sometimes accompanied by dyskinesia. Immunesystem ordinarily acts as a protective mechanism of the living bodyagainst a substance foreign and harmful to the host that invades fromthe environment into the living body. However, the system may sometimesact unfavorably against the living body. When this results inpathological conditions, it is called autoimmune diseases. Diseasesexhibiting dyskinesia due to the autoimmune reaction include, forexample, myasthenia gravis, multiple sclerosis, and rheumatoidarthritis.

Among these, multiple sclerosis, a kind of demyelination diseases, ischaracterized by the presence of a variety of demyelination nests ofvarious sizes dispersed within the white matter of the central nervoussystem with varied lesions of old and new. The lesions arepreferentially observed in the white matter such as the periphery of thelateral ventricle, the optic nerve, the brain stem or the spinal cord.Histologically, it is a disease where oligodendrocytes, engaged information of medullary sheath, are injured and a large number ofoligodendrocytes are observed to have undertaken apoptosis in thelesions. The demyelination nests exhibit at early stage inflammatoryinfiltration of the cells, which are subsequently consolidated uponreplacement with glial fiber at chronic stage. Its clinical symptomsinclude a various combination of symptoms such as optic neuritis,multiple vision, ocular motor disturbance such as nystagmus, spasticparalysis, painful tonic seizure, Lhermitte syndrome, ataxia, mogilaliaand vesicorectal disturbance, where remission is often observed.

In case of cerebrovascular disorders, ischemic conditions in the braininduced by various causes such as cerebral infarction or intracerebralhemorrhage bring about oxygen lack and nutritional disturbance to thecells and tissues in the ischemic region. The oxygen lack first provideswith functional impairment via blockage of ATP production, then leads todegeneration, necrosis or atrophy. Specifically, decrease in oxygen andglucose provisions due to ischemia triggers generation of free radicals,excess accumulation of glutamic acid, increase in intracellular calciumion level, and enhancement of NO production, eventually resulting indeath of neurocytes. The ischemic disturbance depends on the duration ofischemia such that the longer the duration of ischemia is, the moreexacerbated the reperfusion injury becomes. With these reasons, celldeath or cellular degeneration of neurocytes can be the cause ofdyskinesia.

As stated above, neurodegenerative diseases with dyskinesia exhibit celldeath or cellular degeneration of neurocytes as a common pathologyalthough they may include various diseases. Therefore, any substancethat may inhibit or ameliorate cell death or cellular degeneration ofneurocytes would be considered to be an effective medicament fortreating neurodegenerative diseases, especially dyskinesia. At present,for ameliorating these symptoms, a variety of medical drugs have beenproposed, including, for example, a substance that prevents or controlsoxidative disturbance in oxidases in the living body such as superoxidedismutase, catalase, or glutathione peroxidase, as well as clinicallyused medicaments such as trimetazidine hydrochloride, a medicament fortreating ischemic heart disease; sodium oxagrel, thromboxane synthetaseinhibitor; ifenprodil tartrate, a medicament for improving cerebralcirculation and metabolism; and nizofenone fumarate, a medicament forimproving ischemic encephalopathy. However, these medicaments cannotentirely inhibit dyskinesia.

As such, little medicament is known for effectively amelioratingsymptoms in dyskinesia associated with cell death or cellulardegeneration of neurocytes and hence symptomatic treatment hasprincipally been employed. Thus, for instance, a medicament for treatingParkinson disease is used for parkinsonism such as tremor in hands, oran autonomic drug for autonomic symptoms such as orthostatichypotension. Accordingly, for ameliorating dyskinesia induced by celldeath or cellular degeneration of neurocytes, a substance is desiredthat can inhibit cell death or cellular degeneration generated underdisadvantageous conditions for the neurocytes, especially a substancethat can elevate anti-oxidative capacity of the cells.

The explanation mentioned above is described by reference to “Why cellsdie due to ischemia” by Kunio Tagawa, Kyoritsu Publishers Co., Ltd.;“Handbook of stroke” by Keiji Sano, I. P. C.; “Current Diagnosis andTreatment”, CD-ROM, Igaku-Shoin Ltd.; “Grand Medical Dictionary”,CD-ROM, Nanzando Co., Ltd.; “Up-to-date Grand Medical Dicitionary”,CD-ROM, 2nd Ed., Ishiyaku Publishers, Inc.; and “Apoptosis andDiseases”, Ed. by Yoshikuni Mizuno, Iyaku (Medicine and Drug) JournalCo., Ltd.

DISCLOSURE OF THE INVENTION

Under the circumstances, the present inventors have found thatselenoprotein P, a protein derived from blood components, morepreferably a peptide fragment from the C-terminal of selenoprotein P,exhibits a cell death-inhibitory activity, which hitherto has not beenreported, and have filed a patent application (PCT/JP99/06322) for thisfinding. The present inventors further investigated for providing anovel medicament for ameliorating neurodegenerative diseases, especiallyfor ameliorating dyskinesia. As a result, selenoprotein P or a peptidefragment or a series of peptide fragments derived from the C-terminal ofselenoprotein P surprisingly proved to be efficacious as a medicamentfor ameliorating dyskinesia in humans or other animals as demonstratedin animal models which received in vivo administration thereof. Based onthis finding, the present inventors have thus completed the presentinvention.

That is, the present invention relates to a medicament for amelioratingdyskinesia comprising as an active ingredient selenoprotein P and/or apeptide fragment or a series of peptide fragments derived from theC-terminal of selenoprotein P.

In a preferable embodiment of the present invention, the peptidefragment or a series of the peptide fragments from the C-terminal ofselenoprotein P is one having the amino acid sequence consisting of 103amino acid residues from the C-terminal of selenoprotein P, or havingsaid amino acid sequence with one or several amino acid residues thereinbeing deleted, substituted or added, or having a partial sequence ofeither of the above amino acid sequences, or having any of the aboveamino acid sequences as a part of a whole sequence.

In a more preferable embodiment of the present invention, the peptidefragment or a series of the peptide fragments from the C-terminal ofselenoprotein P has the amino acid sequences of the formula (I): Lys ArgCys Ile Asn Gin Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro ArgSer Xaa Cys Cys His Cys Arg His Leu (SEQ ID NO: 1) and/or the formula(II): Thr Gly Ser Ala Ile Thr Xaa Gin Cys Lys Glu Asn Leu Pro Ser LeuCys Ser Xaa Gin Gly Leu Arg Ala Glu Glu Asn Ile (SEQ ID NO: 2)

wherein Xaa represents selenocysteine, or said amino acid sequences withone or several amino acid residues therein being deleted, substituted oradded, or a partial sequence of either of the above amino acidsequences, or an amino acid sequence comprising as a part any of theabove amino acid sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an effect of selenoprotein P to ameliorate dyskinesiainduced by blocking both carotid arteries in gerbil mice.

FIG. 2 shows an effect of selenoprotein P to ameliorate dyskinesia inmouse EAE.

FIG. 3 is a microscopic photograph of a human neurocyte culture with(SeP(+)) or without (SeP(−)) selenoprotein P. When selenoprotein P isadded, protrusion formation was observed in cultured human neurocytes.

BEST MODE FOR CARRYING OUT THE INVENTION

Selenoprotein P was identified in 1977 as a selenium-containing proteinother than glutathione-peroxidase. In 1982, it was revealed thatselenium was incorporated into said protein in the form ofselenocysteine. Moreover, in 1991, a full-length amino acid sequence ofselenoprotein P was determined by cloning selenoprotein P cDNA and, as aresult, possibility that said protein contains at most tenselenocysteine residues was demonstrated (Hill K. E. and Burk R. F.,Biomed. Environ. Sci., 10, p. 198-208 (1997)). Little was known aboutthe function of selenoprotein P. However, it has recently beendemonstrated that selenoprotein P exhibits an activity to reducephospholipid hydroperoxide or peroxynitrite in vitro and acts as asurvival promoting factor of neurocytes.

As demonstrated in the Examples hereinbelow, selenoprotein P proved tohave an activity to ameliorate dyskinesia as it markedly recoveredcapacity of locomotion in Klotho mice, a mouse model of aging, and alsoexhibited an effect to inhibit onset of multiple sclerosis as well as todecrease symptoms of said disease in an experimental allergicencephalomyelitis (EAE), a model of multiple sclerosis. It was alsoshown that selenoprotein P was efficacious for neurological symptomsafter ischemia/reperfusion, especially dyskinesia, in an experiment ofboth carotid arteries blocking in gerbil mice, a model ofcerebrovascular disorders. Moreover, it was revealed that selenoproteinP could form protrusions in cultured neurocytes to thereby recoverfunctions of injured cells. It was thus demonstrated that selenoproteinP had an activity to ameliorate neurological disturbances, especiallysymptoms of dyskinesia, irrespective of their causes, includingischemia/reperfusion injury.

The present invention relates to a novel pharmaceutical efficacy ofselenoprotein P based on the new findings as mentioned above and anactive ingredient of a medicament for ameliorating neuropathy of thepresent invention is selenoprotein P. More specifically, selenocysteine,a selenium-containing amino acid, contained in selenoprotein P isthought to be responsible for amelioration of neuropathy, especiallydyskinesia. The present inventors have disclosed in the above patentapplication that a peptide fragment derived from the C-terminal ofselenoprotein P, a protein from blood components, exhibited a celldeath-inhibitory activity, which hitherto has not been reported.Selenocysteine contained in selenoprotein P is apparently involved inthis activity. Hence, a protein and/or a series of peptides thatcontains selenocysteine and has a cell death-inhibitory activity can bea candidate of a medicament for ameliorating dyskinesia.

Selenium per se, as involved in the present invention, is one ofessential trace elements and it is known that deficiency thereof inducesa serious deficiency disease accompanied by, for instance,cardiomyopathy. It is also demonstrated that selenium is essential forsurvival, maintenance of life or growth of cells as can be seen fromthat addition of sodium selenite to culture medium is indispensableduring serum-free culture. However, as will be understood from the factthat selenium compounds are designated as poisonous substance, adifference between effective and toxic amounts, i.e. a safety range ofconcentration, is small and hence selenium compounds used in an excessamount may be toxic to cells to induce unfavorably cell death. Acutetoxic symptoms of selenium include, for example, pale face, neurologicalsymptoms, dermatitis, and gastrointestinal disorders. It is also knownthat selenocystine, a dimer of selenocysteine, exhibits fairly strongtoxicity when added alone to cell culture. On the contrary, no strongtoxicity was observed in selenoprotein P or a peptide fragment derivedfrom the C-terminal of selenoprotein P according to the presentinvention in spite of the presence of 9 to 10 selenocysteines therein.From this, selenoprotein P with the pharmaceutical efficacy according tothe present invention is characteristic in that it not only containsselenocysteine but also possesses reduced toxicity. Thus, a peptide or aseries of peptides of the present invention allows for providingselenium compounds that not only have reduced toxicity but also exhibitan unexpected activity to ameliorate dyskinesia.

Selenoprotein P as used herein includes any selenoprotein P in anymolecular type without any restriction as far as it exhibits a desiredactivity to ameliorate dyskinesia, including selenoprotein P as anintact molecule or in any of other various molecule types. Among these,preferred is a peptide fragment or a series of the peptide fragmentsfrom the C-terminal of selenoprotein P. Most preferred is the peptidefragment or a series of the peptide fragments from the C-terminal ofselenoprotein P that has the amino acid sequence consisting of 103 aminoacid residues from the C-terminal of selenoprotein P (260th to 362ndamino acids):

260Lys Arg Cys Ile Asn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu LeuAla Pro Arg Ser Xaa Cys Cys His Cys Arg His Leu Ile Phe Glu Lys Thr GlySer Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys Ser Xaa GlnGly Leu Arg Ala Glu Glu Asn Ile Thr Glu Ser Cys Gln Xaa Arg Leu Pro ProAla Ala. Xaa Gin Ile Ser Gln Gln Leu Ile Pro Thr Glu Ala Ser Ala Ser XaaArg Xaa Lys Asn Gln Ala Lys Lys Xaa Glu Xaa Pro Ser Asn362 (SEQ ID NO:3)

wherein Xaa represents selenocysteine, or said amino acid sequence withone or several amino acid residues therein being deleted, substituted oradded, or a partial sequence of either of the above amino acidsequences, or an amino acid sequence comprising as a part any of theabove amino acid sequences.

The term “a series of the peptide fragments” as used herein refers to agroup of peptide fragments with different minute structures due topresence or absence of glycosylation, difference in electric charge,diversity in fragmentation, etc., each of the peptide fragments beingderived from the amino acid sequence of selenoprotein P, having at leastone selenocysteine, and having said amino acid sequence with one orseveral amino acid residues therein being deleted, substituted or added.That is, selenoprotein P and a series of the peptide fragments accordingto the present invention includes any molecules that are derived fromthe amino acid sequence of selenoprotein P and have acytotoxicity-inhibitory activity, including selenoprotein P as an intactmolecule as well as peptide fragments from the C-terminal ofselenoprotein P. The peptide fragments of the present invention may beprepared by the conventional methods using a peptide synthesizer.Alternatively, chemical compounds may also be designed by using thepeptide fragments of the present invention as a lead substance.

Selenoprotein P or the peptide fragment or a series of the peptidefragments derived from said protein for use in the present invention maybe prepared by any process known in the art, for example, by isolationfrom human blood, or by the genetic recombination technique.Selenoprotein P or the peptide fragment or a series of the peptidefragments derived from said protein for use in the present invention asan active ingredient of a medicament for ameliorating dyskinesia israther stable to heat, a denaturing agent, a broad range of pH orprotease in blood as compared to common enzymes. Thus, for purificationand identification thereof, a wide variety of fractionation proceduresmay be applicable, including, for example, fractionations withapplicable various carriers such as various chromatographic proceduressuch as heparin chromatography, cation exchange chromatography, anionexchange chromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, hydroxyapatitechromatography, or various affinity chromatography such as that with acolumn bound with an antibody, using plasma as a starting material. Inaddition to these, other various fractionations may also be applicablesuch as ammonium sulfate precipitation, molecular size fractionationwith membrane, isoelectric focusing, electrophoretic fractionation, etc.A various combination of these fractionations may suitably be used toeffectively fractionate selenoprotein P or the peptide fragment or aseries of the peptide fragments derived from said protein. An example ofpreferable combinations is shown in Preparation 1.

According to the present invention, selenoprotein P or the peptidefragment or a series of the peptide fragments derived from said proteinas an active ingredient may be combined with a suitable known excipientto produce a medicament for ameliorating dyskinesia. An effective doseof the medicament for ameliorating dyskinesia of the present inventionmay vary depending upon ages of subject, symptoms, severity, etc. andultimately upon discretion of a physician. A pharmaceutical efficacydoes not depend upon a route of administration but subcutaneous,intradermal, or intraperitoneal administration, or bolus administrationwithin blood vessels or intravenous drip infusion is much preferred. Itis also possible to administer orally or transdermally in case ofpeptides with a low molecular weight.

A medicament for ameliorating dyskinesia of the present invention maysuitably be applied to any diseases with neurological symptoms,especially with decrease in motor function, induced by cell death orcellular degeneration of neurocytes, including, for instance,cerebrovascular disorders including cerebral infarction, cerebralhemorrhage, subarachnoid hemorrhage, multi-infarct dementia, Binswangertype leukoencephalopathy, and chronic subdural hematoma, ischemicorganopathy, reperfusion injury such as graft of organ, angiopathy,neuropathy, arterial sclerosis, myocardial infarction, autoimmunediseases such as multiple sclerosis, Guillain-Barré syndrome andcollagen disease, neurodegenerative diseases such as spinocerebellardegeneration, Shy-Drager syndrome, Machado-Joseph disease, myotonicdystrophy, Friedreich's syndrome, amyotrophic lateral sclerosis,Alzheimer disease, Pick disease, Huntington chorea, Parkinson disease,progressive supranuclear palsy and epilepsy, dementia or dysbasiaassociated with aging, or traumatic injury of spinal chord or cerebraldisorders due to traffic accident. Among these, it may most preferablybe applied to dyskinesia, epilepsy, motor neuron disorder, disorderassociated with aging, traumatic disorder, cerebrovascular disorder, andimmunopathy. A medicament for ameliorating neuropathy such as dyskinesiaof the present invention comprising as an active ingredientselenoprotein P or a peptide or a series of peptides derived from saidprotein may be administered alone or in combination with other medicaldrug where synergetic effect may be expected. It may effectively beadministered both for the purpose of prevention and treatment.

According to the present invention, a medicament for amelioratingneuropathy for neurodegenerative disorders induced by various causes,especially for disorders with a decrease in motor function.

The present invention is explained in more detail by means of thefollowing Preparation and Examples which are not intended to restrict ascope of the present invention in any sense. Reagents used in thefollowing Preparation and Examples were obtained from Wako Pure ChemicalIndustries, Ltd., TAKARA SHUZO CO., Ltd., Toyobo, New England BioLabs,Pharmacia, BioRad, Sigma and Gibco BRL.

PREPARATION 1 Purification of Selenoprotein P Fragment UsingAnti-Selenoprotein P Fragment Antibody-Bound Carrier (Anti-SeP AntibodyColumn)

Heparin Sepharose-binding fraction from plasma was precipitated with 2 Mammonium sulfate. The precipitate was dissolved in more than 5 volumesof 20 mM Tris buffer, pH 8.0. Selenoprotein P present in this solutionwas adsorbed to anti-SeP antibody column and the carrier was washed withPBS. Selenoprotein P was eluted with 20 mM citrate buffer containing 4 Murea and was adsorbed to a cation exchanger (Macroprep High S: BioRad)equilibrated with 20 mM citrate buffer. Then, gradient elution wasperformed with a salt concentration of sodium chloride and a fraction ofselenoprotein P fragment having the cell death-inhibitory activity wasrecovered. At this stage, a full-length selenoprotein P could also beobtained but with a cell death-inhibitory activity per proteins beingmuch lower than that of the fragment thereof. According to theprocedures as described herein, purification may be carried out in ashort time and hence selenoprotein P fragments with higher celldeath-inhibitory activity per proteins could be obtained. The fragmentsobtained at this stage were also a fraction of a mixture containingvarious molecular species with varied sizes depending on the presence orabsence of glycosylation, intermolecular bonding, or inner cleavage,etc. They were a group of selenoprotein P fragments that showed a sizeranging from 10 to 30 kDa in electrophoresis under non-reductivecondition.

EXAMPLE 1 Cytotoxicity-Inhibitory Activity

Using Dami cells (described in Greenberg S. M. et al., Blood, vol. 72,p. 1968-1977 (1988)) for use in assay system for cytotoxicity-inhibitoryactivity, the cells were washed twice with assay medium (50%PBS/SA/0.03% HSA (manufactured by SIGMA) or SA/0.05% BSA free from fattyacid (WAKO)/4 μM long-chain polyvalent fatty acid (e.g. arachidonicacid, linoleic acid or linolenic acid)) and suspended in the same mediumat 3×10⁴ cells/ml. The cell suspension was added to a 96-well plate ineach 200 μl for wells for sample addition or in each 100 μl for wellsfor serial dilution. To the wells for sample addition was added each 2μl assay sample containing either selenoprotein P fragment prepared inPreparation 1, selenocystine, selenomethionine, Ebselen, or sodiumselenite at the same concentration. After stirring, a serial dilutionwas made with the wells containing 100 μl cell suspension. The plate wasincubated at 37° C. in CO₂ incubator for 4 to 5 days followed byassessment of survival of the cells.

As a result, in addition to selenoprotein P fragments purified inPreparation 1, a peptide purified therefrom under reduced condition (LysArg Cys Ile Asn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala ProArg Ser Xaa Cys Cys His Cys Arg His Leu Ile Phe Glu Lys: SEQ ID NO: 4)proved to exhibit cytotoxicity-inhibitory activity. On the contrary, aselenoprotein P-derived peptide with no selenocysteine (Lys Arg Cys IleAsn Gln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser: SEQID NO: 5) proved to exhibit no cytotoxicity-inhibitory activity,demonstrating that selenocysteine is indispensable for the activity ofselenoprotein P.

EXAMPLE 2 Inhibitory Effect of Selenoprotein P Fragment onIschemia/Reperfusion Injury in Cerebral Ischemia/Reperfusion InjuryModel

Effect of selenoprotein P on dyskinesia induced by cerebralischemia/reperfusion injury was assessed with a degree of paralysisusing gerbil mice of 12 weeks old. The animals were systemicallyanesthetized by intraperitoneal injection of ketamine hydrochloride (100mg/kg), the cervical vein was revealed by midline incision, and received1 mg/animal of selenoprotein P fragment of the present invention viaintravenous administration. After ischemia for 30 minutes or 40 minutes,the bloodstream was recovered for reperfusion. A degree of paralysis wasassessed after 6 and 24 hours.

A degree of paralysis after 6 and 24 hours was assessed in accordancewith the score indicated in Table 1 below. TABLE 1 Normal 0 Lightparalysis in the forefoot, dull movement 1 in a posture of a bent foot Alittle worsened paralysis in the foot, 2 continuously turning round toone side Worsened paralysis, falling down to one side 3 Still worsenedparalysis, incapable of moving 4 Death 5

As shous in FIG. 1, assesing the state of paralysis after 6 and 24 hoursin the 30 minutes ischemia model, the results indicated that aselenoprotein P-receiving group apparently exhibited better conditions.In case of the 45 minutes ischemia model, although no significantdifference in a degree of paralysis was observed after 6 hours, muchdifference could be seen after 24 hours.

EXAPLE 3 Effect of Selenoprotein P Administration on Ataxia in KlothoMice

Effect of selenoprotein P fragment of the present invention onamelioration of decrease in motor function associated with aging wasinvestigated using Klotho mice (Nature, 390: 45-51, 1997). Klotho mouse,an aging model mouse generated by Dr. Yohichi Nabeshima at KyotoUniversity, the postgraduate medical course in 1997, has an average lifespan of 60 days, displays retarded growth at around 20 days and inalmost every case dies within around 100 days after birth. Due to Klothogene mutation, it exhibits various abnormalities related to aging suchas arterial sclerosis, emphysema, atrophoderma, atrophy of the gonad (nomeiosis of ovum and sperm), neurodegeneration (drop-off of Purkinjecells in the cerebellum, ataxia), malfunction of hypophysis (decrease ingrowth hormone production, growth disturbance), and osteoporosis(ectopic calcification in the soft tissue or cartilage) as described inNature, 390: 45-51. 1997. Klotho mice were obtained from CLEA Japan,Inc. with approval of Dr. Yohichi Nabeshima at Kyoto University, thepostgraduate medical course.

Four Klotho mice of 4 weeks old each in two groups received weeklyeither selenoprotein P fragment (1.5 mg/ml) prepared in Preparation 1dissolved in saline (300 μl) or saline alone (300 upl) viaintraperitoneal injection and the conditions were observed up till 8weeks old. As a result, no weight increase was observed in both groupswhere either saline alone or selenoprotein P fragment was administeredand hence pathogenesis could not fundamentally be removed. However, incomparison of their behavior, the mice administered with selenoproteinP, when put on a palm, could jump off spontaneously whereas the miceadministered with saline alone could not spontaneously escape,suggesting that selenoprotein P fragment may effectively amelioratedecrease in motor function.

EXAMPLE 4 Experimental Allergic Encephalomyelitis (EAE) in Mice

For investigating the effect of selenoprotein P on dyskinesia induced byimmunopathy based on a degree of paralysis, an experiment was performedwherein selenoprotein P was administered to EAE mice. FemaleSJL/JOrIICrj of 14 weeks old (Charles River Japan, Inc.) was used as atest animal. Myelin proteolipid protein (PLP) synthetic peptide 139-151(amino acid sequence: His Ser Leu Gly Lys Trp Leu Gly His Pro Asp LysPhe (SEQ ID NO: 6); 25 mg/mouse) as an immunogen was intradermallyadministered to the mice together with Freund complete adjuvant H37Ra(400 mg/mouse) and pertussis toxin (manufactured by List BiologicalLaboratory; 200 ng/mouse) at two portions at the neck of tail.

Thirteen mice each in two groups received daily 0.5 mg/head ofselenoprotein P via intraperitoneal administration from the day (Day 8)previous to onset of the disease till Day 13. A control group receivedsaline via intraperitoneal administration. After onset of the disease, adegree of paralysis was daily assessed in accordance with the scoreindicated in Table 2 below. TABLE 2 Normal 0 Tail hanging downincompletely 0.5 Tail hanging down completely 1 Light paralysis in thehind foot 2 Medium or severe paralysis in the hind foot or 3 lightparalysis in the forefoot Complete paralysis in the hind foot and/or 4medium to severe paralysis in the forefoot Paralysis in the limbs ordying condition 5 Death 6

As shown in FIG. 2, the results indicated that onset of the disease wasinhibited and severity of the disease was lowered in the groupadministered with selenoprotein P. Besides, in comparison of a rate ofonset of the disease on Day 10, 12 among the total 13 mice exhibitedonset of the disease in the group administered with saline whereas only4 among the total 13 mice did in the group administered withselenoprotein P. Thus, selenoprotein P proved to effectively inhibitonset of immunopathy and to ameliorate dyskinesia.

EXAMPLE 5 Effect of Selenoprotein P on Protrusion Formation in CulturedHuman Neurocytes

NT2 (Ntera2/D1) (Pleasure, S. J. and Lee, V. M., 1993, J. Neurosci. Res.35: 585-602) is a progenitor neurocyte derived from humanteratocarcinoma and can be differentiated with retinoic acid into hNTneurocytes having the properties of the nerve in the central nervoussystem (Trojanowski, J. Q. et al., 1993 Exp. Neurol. 122: 283-294).Effect of selenoprotein P on protrusion formation in the hNT cells wasinvestigated.

Both NT2 and hNT cells can be cultured in CO₂ incubator at 37° C. ThehNT cells were prepared as follows: NT2 cells, being capable of passageculture in 10% FCS/D-MEM/F-12 medium, were first plated at 2.5×10⁵cells/ml. After 24 hours, the culture medium was replaced withD-MEM/F-12 containing 10 μM all-trans retinoic acid to initiateinduction. The culture was subsequently continued for six weeks whilereplacing with the same medium every three days to differentiate NT2cells into hNT cells. All the differentiated cells were recovered andexpanded by 3-folds by passage culture with 10% FCS/D-MEM/F-12 medium.After 48 hours, the cells were plated at 3 to 4×10⁶ cells/ml. After48-hour culture, the culture medium was replaced with 5% FCS/D-MEM/F-12plus mitotic inhibitor (10 pM Fluorodeoxyuridine, Uridine, 1 μM cytosinearabinoside) and culture was continued for ten days while replacing withthe same medium every three days. Subsequently, the culture was treatedwith trypsin for a short time period and then lightly tapped to releasethe hNT cells which were recovered. The recovered cells were suspendedin a mixed medium of an equal amount of NT2 culture supernatant (10%FCS/D-MEM/F-12 medium) and a fresh 10% FCS/D-MEM/F-12 medium. Thesuspension was plated on Laminin coating plate to maintain the cells.The hNT cells thus obtained were used in the following experiment.

The hNT cells were recovered by trypsin treatment, washed with a basalmedium of RPMI 1640/D-MEM/F-12 (1:2:2), suspended in the same basalmedium and cultured in 96-well plate at 3000 cells/well. On Day 5 to Day7, the cells died completely. On the contrary, when selenoprotein P wasadded at 100 ng/ml, cell death was completely inhibited to allow thecells to survive for more than two weeks. Besides, cellular morphologywas observed on Day 4 to reveal that protrusion was formed whenselenoprotein P was added as-shown in FIG. 3. Thus, selenoprotein Pproved to effectively induce protrusion formation such as dendrite oraxon in neurocytes.

1. A medicament for ameliorating dyskinesia, comprising as an activeingredient selenoprotein P and/or a peptide fragment or a series ofpeptide fragments derived from the C-terminal of selenoprotein P.
 2. Themedicament for ameliorating dyskinesia according to claim 1 wherein saidpeptide fragment or a series of peptide fragments derived from theC-terminal of selenoprotein P is a peptide or a series of peptides witha cytotoxicity-inhibitory activity having the amino acid sequence from260th to 362nd amino acid residues from the C-terminal of selenoproteinP, or said amino acid sequence with one or several amino acid residuestherein being deleted, substituted or added, or a partial sequence ofeither of the above amino acid sequences, or an amino acid sequencecomprising as a part any of the above amino acid sequences.
 3. Themedicament for ameliorating dyskinesia according to claim 1 or 2 whereinsaid peptide fragment or a series of peptide fragments derived from theC-terminal of selenoprotein P is a peptide or a series of peptideshaving the amino acid sequences of the formula (I): Lys Arg Cys Ile AsnGln Leu Leu Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Xaa CysCys His Cys Arg His Leu (SEQ ID NO: 1) and/or the formula (II): Thr GlySer Ala Ile Thr Xaa Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys Ser Xaa GlnGly Leu Arg Ala Glu Glu Asn Ile (SEQ ID NO: 2). wherein Xaa representsselenocysteine, or said amino acid sequences with one or several aminoacid residues therein being deleted, substituted or added, or a partialsequence of either of the above amino acid sequences, or an amino acidsequence comprising as a part any of the above amino acid sequences. 4.The medicament for ameliorating dyskinesia according to claim 1 whereinsaid dyskinesia is one associated with neurodegenerative diseases. 5.The medicament for ameliorating dyskinesia according to claim 1 whereinsaid dyskinesia is one associated with aging, traumatic neuropathy,cerebrovascular disorders or immunopathy.
 6. A medicament for treatingneurodegenerative diseases, comprising as an active ingredientselenoprotein P and/or a peptide fragment or a series of peptidefragments derived from the C-terminal of selenoprotein P.
 7. Themedicament for treating neurodegenerative diseases according to claim 6wherein said neurodegenerative diseases are selected from ataxia,epilepsy, motor neuron disorder, aging, traumatic neuropathy,cerebrovascular disorders or immunopathy.